The present invention relates to an integrated-chip-type biosensor and a related method for detection of pathogenic substances. The biosensor and method are particularly, but not exclusively, useful-in detecting foodborne pathogens such as Listeria monocytogenes. 
Pathogenic bacteria in foods are the cause of 90% of the cases of reported foodborne illnesses. The Centers for Disease Control and Prevention estimate that there 76 million cases of foodborne illnesses each year in the United States, resulting in hospitalization of 325,000 people, 5,500 deaths, and an annular cost of $7 to $23 billion. E. coli O157: H7 and Listeria monocytogenes are the pathogens of most concern. Ground meat containing E. coli O157: H7 is now considered to be an adulterated food while Listeria monocytogenes has emerged as one of the most important food pathogens with a xe2x80x9czero tolerancexe2x80x9d criterion for it in ready-to-eat processed (lunch) meats and dairy foods.
The genus Listeria is comprised of six species, L. monocytogenes, L. ivanovii, L. seeligeri, L. innocua, L. welshimeri, and L. grayi. Of these species, only L. monocytogenes is harmful to humans. Consumption of contaminated food may cause meningitis, encephalitis, liver abscess, headache, fever and gastroenteritis (diarrhea) in immunologically challenged individuals and abortion in pregnant women. L. monocytogenes is ubiquitous in nature and can be found in meat, poultry, seafood, and vegetables. Occurrence of this organism could be as high as 32%. In a food sample, L. monocytogenes is often present in close association with other nonpathogenic Listeria species, thereby complicating the specific detection procedures. A successful detection method ideally detects only L. monocytogenes in the presence of overwhelming populations of nonpathogenic Listeria and other background resident bacteria.
The food processing industry annually carries out more than 144 million microbial tests costing $5 to $10 each. About 24 million of these tests are for detection of food pathogens based on biochemical profile analysis, immunogenic tests (such as enzyme linked immuno-sorbent assays or ELISA), and DNA/RNA probes. These tests are reliable but most require two to seven days to complete because of the steps that are needed to resuscitate cells, increase cell numbers or amplify genetic material needed for detection. This time period is too long for real-time detection of contamination in a food plant and is sufficiently long for contaminated food to be formulated, processed, packaged, shipped, and purchased and eaten by the consumer. Current tests require at least several days to confirm presence of Listeria monocytogenes. The number of annual tests is only expected to increase due to heightened consumer concerns about food safety and the requirement of compulsory testing.
In general, diagnostic tools used for detecting or quantitating biological analytes rely on ligand-specific binding between a ligand and a receptor. Ligand/receptor binding pairs used commonly in diagnostics include antigen-antibody, hormone-receptor, drug-receptor, cell surface antigen-lectin, biotin-avidin, substrate/enzyme, and complementary nucleic acid strands. The analyte to be detected may be either member of the binding pair; alternatively, the analyte may be a ligand analog that competes with the ligand for binding to the complement receptor.
A variety of devices for detecting ligand/receptor interactions are known. The most basic of these are purely chemical/enzymatic assays in which the presence or amount of analyte is detected by measuring or quantitating a detectable reaction product, such as a detectable marker or reporter molecule or ligand. Ligand/receptor interactions can also be detected and quantitated by radiolabel assays.
Quantitative binding assays of this type involve two separate components: a reaction substrate, e.g., a solid-phase test strip and a separate reader or detector device, such as a scintillation counter or spectrophotometer. The substrate is generally unsuited to multiple assays, or to miniaturization, for handling multiple analyte assays from a small amount of body-fluid sample.
In recent years, there has been a merger of microelectronics and biological sciences to develop what are called xe2x80x9cbiochips.xe2x80x9d The term xe2x80x9cbiochipxe2x80x9d has been used in various contexts but can be defined as a xe2x80x9cmicrofabricated device that is used for delivery, processing, and analysis of biological species (molecules, cells, etc.).xe2x80x9d Such devices have been used, among other things, for the direct interrogation of the electric properties and behavior of cells (Borkholder et al. xe2x80x9cPlanar Electrode Array Systems for Neural Recording and Impedance Measurementsxe2x80x9d, IEEE Journal of Microelectromechanical Systems, vol 8(1), pp. 50-57, 1999); impedance-based detection of protein binding to surfaces, antigen-antibody binding, and DNA hybridization (DeSilva et al., xe2x80x9cImpedance Based Sensing of the Specific Binding Reaction Staphylococcus Enterotoxin B and its Antibody on an Ultra-thin Platinum Film,xe2x80x9d Biosensors and Bioelectronics, vol. B 44, pp 578-584, 1995); micro-scale capillary electrophoresis (Wooley et al., :Ultra High Speed DNA Sequencing Using Capillary Electrophoresis Chips,xe2x80x9d Analytical Chemistry, vol. 67(20), pp. 3676-3680, 1995); and optical detection of DNA hybridization using fluorescence signals in the commercially available xe2x80x9cDNA-chipsxe2x80x9d (Fodor et al., xe2x80x9cLight-directed Spatially Addressable Parallel Chemical Synthesis,xe2x80x9d Science, vol. 251, pp. 767-773).
One of the most interesting uses of biochips is for the detection of small quantities of pathogenic bacteria or toxigenic substances in food, bodily fluids, tissue samples, soil, etc. In applications such as the screening of food products for the presence of pathogenic bacteria, it would be beneficial to detect between 100 and 1000 microorganisms per milliliter of sample, with a sample volume of a couple of milliliters. Not counting the fact that bacteria are substantially larger than single biomolecules (xcx9c2 xcexcm vs. xcx9c10-100 xc3x85), 1000 cells are approximately equivalent to a 10xe2x88x925 femto-moles of cells, which gives an idea of the difficulty in directly detecting such a small number suspended in a volume of 1 or 2 ml, along with large numbers of food debris, proteins, carbohydrates, oils, and other bacteria. Additionally, in many cases the screening technique must be able to discern between viable and dead cells. Many bacteria will not produce toxins when not viable and consequently will not be pathogenic in that state. DNA detection methods, which search for DNA sequences specific to the pathogen of interest, can be extremely sensitive because they rely on the very specific binding of complementary DNA strands, often coupled with Polymerase Chain Reaction (PCR) for amplification. But the detected DNA fragments cannot reveal whether the pathogen was viable or not. These are the main reasons why current methods of detection almost always involve a growth step, in which the bacteria are cultured to increase their numbers by several orders of magnitude. Once the bacteria are amplified to a large number, visual detection of colonies or Enzyme-Linked Immunosorbent Assays (ELISA) confirm their presence in the original sample. Even though bacteria can multiply very rapidly, this amplification by means of extended growth makes conventional detection methods extremely lengthy, taking anywhere from 2 to 7 days. Thus, one of the main goals of micro-scale detection is a reduced time of analysis, on the order of 2 to 4 hours, to be better than the more conventional methods like plate counts and ELISA.
Numerous reports can be found in the literature on biosensors based on the impedimetric detection of biological binding events, or the amperometric detection of enzymatic reactions. (See DeSilva et al., xe2x80x9cImpedance Based Sensing of the Specific Binding Reaction Staphylococcus Enterotoxin B and its Antibody on an Ultra-thin Platinum Film,xe2x80x9d Biosensors and Bioelectronics, vol. B 44, pp 578-584, 1995; Mirsky et al., xe2x80x9cCapacitive Monitoring of Protein Immobilization and Antigen-antibody Reactions on Monomolecular Alkylthiol films on Gold Electrodes,xe2x80x9d Biosensors and Bioelectronics, vol. 112(9-10), pp. 977-989, 1997; Berggren et al., xe2x80x9cAn Immunilogical Interleukine-6 Capacitive Biosensor Using Perturbation with a Potentiostatic Step,xe2x80x9d Biosensors and Bioelectronics, vol. 13, pp. 1061-1068, 1998; Van Gerwen et al., xe2x80x9cNanoscaled Impedimetric Sensors for Multiparameter Testing of Biochemical Samples,xe2x80x9d Sensors and Actuators, vol. B 49, pp. 73-80, 1998; Hoshi et al., xe2x80x9cElectrochemical Deposition of Avidin on the Surface of a Platinum Electrode for Enzyme Sensor Applications,xe2x80x9d Analytical Chimica Acta, vol. 289, pp. 321-327, 1994; Jobst et al., xe2x80x9cMass producible Miniaturized Flow Through a Device with a Biosensor Array,xe2x80x9d Sensors and Actuators, vol. B 43, pp. 121-125, 1997; Towe et al., xe2x80x9cA Microflow Amperometric Glucose Biosensor,xe2x80x9d Biosensors and Bioelectronics, vol. 97(9), pp. 893-899, 1997.) Impedimetric detection works by measuring impedance changes produced by the binding of target molecules to receptors (antibodies, for example) immobilized on the surface of microelectrodes. Amperometric devices measure the current generated by electrochemical reactions at the surfaces of microelectrodes, which are commonly coated with enzymes. Both of these methods can be very sensitive, but preparation of the surfaces of the electrodes (immobilization of antibodies or enzymes) is a complex and sometimes unreliable process, they can be prone to drift, and tend to be very sensitive to noise produced by the multitude of species present in real samples (bodily fluids, food, soil, etc.).
Most, if not all, of the above-mentioned devices are not fully integrated biochips, and sometimes lack integrated electrodes and a sealed fluidic path for the injection and extraction of samples. The most common design of these sensors uses thin metal rods or wires as electrodes, immersed in a flow-through cell. And even those devices based on microfabricated biochips either have a fluidic system separately fabricated over the chip, or the samples are dropped over an open reservoir on the chip, or the whole chip is immersed in a vessel containing the fluids. Having a fully closed system permits the incorporation of sample pre-processing steps, like filtering and chromatography, onto the same chip as the detector.
As mentioned earlier, one of the main goals of bacterial sensors is to determine whether the bacterium of interest is indeed live or dead. A technique that has been widely reported to detect the viability of bacteria on a macro-scale relies on measuring the conductance/impedance changes of a medium in which the microbes are cultured. Such a method is recognized by the Association of Official Analytical Chemists International (AOAC) as a standard technique for the detection of Salmonella in food. This is possible because bacterial metabolism changes the electrolyte concentration in the suspension medium, significantly altering the electrical characteristics of the medium.
It is a general object of the present invention to provide a method and/or an associated apparatus for detecting whether a microbiological substance is present in a fluid sample.
A more specific object of the present invention is to provide a method and/or an associated device for a more rapid detection of foodborne pathogens, particularly including, but not necessarily limited to, Listeria monocytogenes. 
An even more specific object of the present invention is to provide such a method and/or device which detects pathogens in a few hours or less, possibly within minutes.
A further specific object of the present invention is to provide such a method and/or device which is capable of detecting a relatively small number of instances of a pathogen such as a bacterium.
Another specific object of the present invention is to provide such a method and/or device which is able to distinguish between a sample of live bacteria and a sample of dead bacteria of the same type.
Another object of the present invention is to provide a method for manufacturing a biosensor, particularly a microscale biosensor.
These and other objects of the present invention will be apparent from the drawings and descriptions herein. Every object of the invention is considered to be attained by at least one embodiment of the invention. However, no embodiment necessarily meets every object set forth herein.
The present invention is directed in part to a microscale biosensor for use in the detection of target biological substances including molecules and cells. A preferred embodiment of a biosensor pursuant to the present invention is a microfluidic system with integrated electronics, inlet-outlet ports and interface schemes, high sensitivity detection of pathogen specificity, and processing of biological materials at semiconductor interfaces.
The present invention is also directed in part to a fabrication process for a microfluidic biochip that is used for impedance spectroscopy of biological species. Key features of the device include an all top-side processing for the formation of fluidic channels, planar fluidic interface ports, integrated metal electrodes for impedance measurements, and a glass cover sealing the non-planar topography of the chip using spin-on-glass as an intermediate bonding layer. In one embodiment of the biosensor chip, the total volume of the fluidic path in the device is on the order of 30 nl.
A method in accordance with the present invention for detecting a microbiological substance utilizes a microfabricated biosensor chip including integrated detection elements. The method comprises delivering a fluid sample to the biosensor chip and thereafter separating at least some contaminants or debris from the fluid sample to at least partially isolate and retain instances of a predetermined target type of microbiological material, a material to be detected, on the biosensor chip. The separating of the contaminants takes place at least in part on the biosensor chip itself. After the separating of contaminants from the fluid sample, the detection elements are operated to determine whether the separated fluid sample contains microbiological material of the predetermined target type.
This method may further comprise carrying out a bioseparations process on the fluid sample prior to the delivering of the fluid sample to the biosensor chip. In accordance with one embodiment of the present invention, the bioseparations process includes adding to the fluid sample a plurality of microscopic carrier elements each provided with a multiplicity of binding agents for coupling the microbiological material to the carrier elements. These carrier elements preferably take the form of beads or microspheres. The separating of contaminants from the fluid sample on the biosensor chip preferably includes trapping the carrier elements with the coupled microbiological material in a detection chamber on the biosensor chip while flushing remaining portions of the fluid sample from the chamber. This trapping of the carrier elements with the coupled microbiological material in a detection chamber serves in part to concentrate the microbiological material of interest and thus enhance the sensitivity of the detection technique. The trapping of the carrier elements may be implemented in part by providing a filter barrier or retention structure at an outlet of the detection chamber. Such a barrier or retention structure preferably takes the form of a microfabricated filter grid or post array. Alternatively, the trapping of the carrier elements, where the carrier elements are made of a magnetic material, in a magnetic field generated in the detection chamber.
In accordance with another, more particular, feature of the present invention, the bioseparations process includes subjecting a the fluid sample (prior to delivery to the biosensor chip, to a bioactive surface taken from the group consisting of a cation exchange resin and an anion exchange resin. The cation exchange resin may include Amberlyst 35 while the anion exchange resin includes IRA 400.
The present invention is especially effective in detecting microbiological material in the form of a pathogenic strain of bacteria such as Listeria monocytogenes. In that case, the methodology includes extracting the fluid sample from a food product prior to delivering of the fluid sample to the biosensor chip. As discussed below, the detection of Listeria monocytogenes is implemented in part by attaching antibodies to a capture surface in the detection chamber of the biosensor. That capture surface may be on an electrode or oxide surface in the detection chamber. Alternatively, the capture surface may be on a bead or microsphere floating in the detection chamber. It will be apparent to one of ordinary skill in the art that virtually any microorganism may be detected by the method of the present invention simply by attaching an appropriate antibody to a capture surface as described herein. Antibodies and their associated antigens on the cell membranes of various microorganisms are well documented in the art. It will also be apparent to one skilled in the art that species other than bacteria may be detected by the methodology of the present invention. Various proteins, peptide groups, nucleic acid chains, and other molecules may be detected by the selection of suitable binding agents and the attachment of those binding agents to a capture surface in a detection chamber of a biosensor.
A biosensor in accordance with the present invention comprises a substrate microfabricated to include, as integrated components, a detection chamber, a first channel segment extending to an inlet of the detection chamber, a second channel segment extending from an outlet of the chamber, and a retention structure for holding, in the detection chamber, a carrier element entraining a target microbiological species and for permitting the passage from the detection chamber of contaminants or debris in a fluid sample containing the carrier element and the target microbiological species. The retention structure may take the form of a filter grid or grating disposed on the substrate on an upstream side of the outlet. Alternatively or additionally, where the carrier element is made of magnetic material, the retention structure may include a magnetic field generating element such as an electromagnet.
The retention structure on the biosensor enables the concentration of a target microbiological species at the point of measurement. This facilitates and enhances the detection process. The small size of the detection chamber, less than 100 microliters and preferably between about 1 picoliter and 1 microliter, also increases the sensitivity of the detection process. Yet another factor contributing to the efficacy of the present methodology is the use of a low conductivity buffer as the fluid matrix in which the microbiological species of interest is entrained in the detection chamber.
The detection chamber is provided with at least one pair of electrodes, preferably with interdigitated finger parts, and has a volume of less than approximately one microliter. The volume of a fluid sample in the device may be substantially less than one microliter, even down to about 1 picoliter. The electrodes are spaced from each other by 1 to 100 microns and, more preferably, by 2 to 50 microns.
A biosensor in accordance with another embodiment of the present invention comprises a substrate microfabricated to include, as integrated components, a detection chamber and a channel extending to an inlet of the detection chamber. The biosensor further comprises a wicking element connected at one end to the substrate so as to be in communication with the channel, for drawing a fluid sample by capillary action to the channel for delivery to the detection chamber. The wicking element may be attached at the one end by an adhesive to the substrate. Where the substrate is microfabricated to include an inlet groove or trench substantially coplanar with the channel and the detection chamber, the one end of the wicking element is disposed in the inlet groove or trench, so that the wicking element is coplanar at the one end with the channel and the detection chamber.
An integrated microscale biosensor in accordance with a further embodiment of the present invention comprises a substrate microfabricated to include, as integrated components, a detection chamber, a channel extending to an inlet of the detection chamber, and an inlet groove or trench substantially coplanar with the channel and the detection chamber. The biosensor further comprises an elongate fluid delivery member having a downstream end disposed in the inlet groove or trench. The fluid delivery member is connected at the downstream end to inlet groove or trench so that at least the downstream end of the fluid delivery member is coplanar with the channel and the detection chamber. The elongate fluid delivery member may take the form of a microbore tube or a wicking element.
Preferably a biosensor chip in accordance with the present invention is top-side processed only. In addition, there is no processing (e.g., cutting) of a cover plate. This structure facilitates the manufacturing process, in part by obviating alignment requirements between the cover plate and the substrate. Thus, the cover attached to the substrate over the detection chamber, the channel, the inlet groove, and the downstream end of the fluid delivery member can be an integral or continuous member, devoid of holes or apertures. Such holes or apertures would be required, for instance, where a feed tube was to be inserted through the cover.
A method for manufacturing a biosensor comprises, in accordance with the present invention, providing a substrate, processing the substrate to generate a detection chamber and a channel extending to the detection chamber, further processing the substrate to provide at least one pair of electrodes in the detection chamber, and exposing the processed substrate to BSA (bovine serum albumin) and avidin to adsorb the avidin to the electrodes in the presence of the BSA.
This manufacturing method may further comprise subjecting the exposed processed substrate to a fluid containing a biotinylated antibody specific to a preselected antigen, thereby attaching the antibody to the electrodes via a biotin-avidin link. In a particular embodiment of the invention, the biotinylated antibody is specific to an antigen on a cell membrane of Listeria monocytogenes. Monoclonal antibody producing clones of C11E9 and EM-7G1 (producing antibodies specific for Listeria monocytogenes) are cultured in growth media in a growth chamber. Antibodies are harvested from culture supernatants by salt (ammonium sulfate) precipitation. After an initial concentration step, carried out by known techniques, high quality antibodies are obtained by further purification through size exclusion chromatography followed by protein-A affinity chromatography in an FPLC system.
A method for manufacturing a biosensor comprises, pursuant to another embodiment of the present invention, processing a substrate to create a shallow detection chamber and a channel extending to the detection chamber, thereafter further processing the substrate to deposit at least one pair of electrodes in the detection chamber, and subsequently processing the substrate to create at least deep groove at a periphery of the substrate, for receiving an elongate fluid delivery element, the channel communicating with the deep groove. A downstream end of the fluid delivery element is inserted into and attached to the deep groove.
This method may further comprise attaching a cover to the substrate over the detection chamber, the channel, the deep groove and the downstream end of the fluid delivery element. Where the cover is made of glass, the attaching of the cover to the substrate includes placing a spin-on-glass composition on the glass, subsequently contacting the substrate with the spin-on-glass composition, and heating the substrate, the cover, and the spin-on-glass composition to enabling a flow of the spin-on-glass composition into interstitial spaces on the substrate and form a fluid-tight seal.
A method for detecting a microorganism comprises, in accordance with the present invention, preparing a fluid sample containing at least one microorganism of a preselected type, the fluid sample having a buffer of a low conductivity liquid, the fluid sample also containing a nonionic nutrient. The fluid sample is disposed in or delivered to a detection chamber having a volume between about 1 picoliter and approximately 1 microliter. The fluid sample is maintained at a predetermined temperature in the detection chamber and an electrical parameter of an electrical circuit incorporating the detection chamber and the fluid sample therein is measured. The electrical parameter is an impedance measure taken from the group consisting of a magnitude and phase. The method is effective in the detection of living Listeria monocytogenes cells. The buffer may be a low conductivity Tris-Glycine buffer.
In accordance with another feature of the present invention, the measuring of the electrical parameter includes measuring the impedance parameter at a plurality of frequencies within a range from 100 Hz to 1 MHz.
A method for testing a food product for the presence of a predetermined type of pathogenic bacteria comprises, in accordance with the present invention, extracting a fluid sample from the food product, feeding the extracted fluid sample providing an integrated microscale biosensor, subjecting the fluid sample to a bioseparations process to remove extraneous particles including proteins and kinds of bacteria other than the predetermined type of pathogenic bacteria, binding bacteria of the predetermined type in the fluid sample to at least one substrate body, and, after the feeding of the extracted fluid sample to the chamber, the subjecting of the fluid sample to the bioseparations process, and the binding of the predetermined type of bacteria to the at least one substrate body, measuring an electrical parameter of an electrical circuit incorporating the detection chamber and the fluid sample therein to detect the presence in the fluid sample of living instances of the predetermined type of bacteria. The binding of the predetermined type of bacteria may be to beads or microspheres floating in the fluid sample. Alternatively or additionally, the binding of the predetermined type of bacteria may be to electrodes in the biosensor. Subjecting of the fluid sample to the bioseparations process may take place at least partially after feeding of the fluid sample to the biosensor.
The sensitivity to biological pathogens of a biosensor chip in accordance with the present invention is based on the placement of protein receptors, derived through biotechnology processes, on a surface of the biosensor. A tiny amount of fluid taken from a specimen such as a processed meat or dairy product is then delivered to the biosensor. If a target bacterium such as Listeria monocytogenes is present, it will bind to the receptor and cause a measurable electronic signal to be generated in no more than several hours and possibly within minutes.
The present invention provides a method and an associated device for the relatively rapid detection of biological pathogens such as bacteria. The method and device can detect small numbers of bacteria such as Listeria monocytogenes in time intervals short enough to enable removal of contaminated products from the stream of commerce before consumption of the products by individuals.
Biosensors or biosensors as disclosed herein improve the quality of life by providing cost-effective means for probing biological materials for pathogenic organisms and molecules in manufacturing facilities, the environment, hospitals, doctors"" offices, and ultimately in the home.
The present invention provides a method and an associated device for a relatively rapid detection of foodborne pathogens. The present invention obviates the time-consuming steps of culturing and transferring cells, if present, to increase their numbers or genetic material to the detectible levels required by conventional detection techniques.
The vast majority of the bacterial detection methods currently in use are based on fluorescent tagging of the bacteria, or on the detection of DNA fragments from the bacterial genome. Both techniques are unable to determine if the microorganism was dead or alive in the original sample, and both require extensive manipulations of the sample. Moreover, any fluorescence technique requires bulky and expensive optical apparatuses for excitation and detection of the fluorescence. Additionally, when the microorganism is present in very small concentrations (10 to 1000 cells per milliliter) a growth step is necessary to increase the concentration, but this can drive the total assay time to anywhere from 2 to 7 days.
The present technique solves some of these problems. By its very nature, the present methodology inherently detects only live microorganisms, which is very important for certain applications, especially in food safety (many microorganisms present in food are not pathogenic if they are dead). The method of the present invention also relies exclusively on electrical signals, making the related equipment less expensive and smaller than others. Additionally, the absence of a lengthy growth step makes detection possible in a couple of hours instead of days.
Instruments for the analysis of the conductivity or impedance of an incubated bacterial suspension have been available for a number of years, but they suffer from two limitations. First, their selectivity is very poor because they rely on the composition of the growth medium for encouraging the proliferation of the microorganism of interest, while suppressing the proliferation of others. The second limitation is related to the scale in which the assay is performed. The available equipment uses volumes of bacterial suspension in the milliliter range and above, which requires large numbers of bacteria to provide a discernible signal. The method of the present invention eliminates the first limitation by selectively capturing the bacteria using antibodies prior to the measurement, and increases the sensitivity for very small numbers of microorganisms (1 to 1000) by confining them to an extremely small volume (1 picoliter to 1 microliter). Additionally, the method of the present invention uses a low conductivity buffer, which increases even further the sensitivity. Even very small amounts of ions released by the microorganisms can produce a large change in impedance (in relative terms), since the ionic concentration of the low conductivity buffer is very low.